1. Field of the Invention
The present invention relates generally to the fields of protein chemistry, endocrinology and gene therapy. More specifically, the present invention relates to a method for production of functional proteins in culture in response to shear stress using a rotating wall vessel.
2. Description of the Related Art
A successful and documented modality to induce polarization and differentiation of cells in culture is the rotating wall vessel (1-4). In rotating wall vessels gravity is balanced by equal and opposite physical forces including shear stresses. In engineering terms this has been claimed to simulated microgravity at boundary conditions [Wolf D. A. and R. P. Schwarz. (1991) NASA Technical Paper 3143].
Rotating wall vessels, including models with perfusion, are a quantum advance. The rotating wall vessel is a horizontally rotated cylindrical cell culture device with a coaxial tubular oxygenator (1, 5-7). The rotating wall vessel induces expression of select tissue-specific proteins in diverse cell cultures (1-2, 8-9). Examples of expression of tissue-specific proteins include carcinoembryonic antigen expression in MIP-101 colon carcinoma cells (2), prostate specific antigen induction in human prostate fibroblasts (7), through matrix material induction during chondrocyte culture (8). The quiescent cell culture environment of the rotating wall vessel balances gravity with shear and other forces without obvious mass transfer tradeoff (1-2, 4). The rotating wall vessel provides a culture environment suitable for co-cultures of diverse cell types, and three dimensional tissue construct formation.
Rotating wall vessel technology is being used in clinical medical practice recently by facilitating pancreatic islet implantation (4, 10). Pancreatic islets are prepared in rotating wall vessels to maintain production and regulation of insulin secretion. The islets are alginate encapsulated to create a non-inflammatory immune haven, and are implanted into the peritoneal cavity of Type I diabetic patients. This implantation of pancreatic islets has maintained normoglycemia for 18 months in diabetic patients, and progressed to Phase III clinical trials (4, 10). These vessels have also been applied to, for example, mammalian skeletal muscle tissue, cartilage, salivary glands, ovarian tumor cells, and colon crypt cells (11-13). Previous studies on shear stress response in endothelial cells, and rotating wall vessel culture have been limited to structural genes (14-16). These studies did not address the issue of a process for the production of functional molecules, such as hormones. Shear stress response elements have not previously been demonstrated in epithelial cells, either for structural genes of production of functional molecules.
Vitamin D dependent rickets has been a disease familiar to family farms and larger animal husbandry industries for centuries (17-18). The development of renal replacement therapy by dialysis in humans expanded vitamin D deficient bone disease from an occasional human clinical caveat to a common clinical problem. This led to identification of the active form of vitamin D as 1, 25-diOH D3 and the development of a multi-billion dollar per year worldwide market, predominantly in end-stage renal disease patients, to provide replacement hormone clinically (18). The active 1,25-diOH form of vitamin D3 is mainly used to treat bone disease in dialysis patients but has *also been implicated as a therapy for osteoporosis, and some forms of cancer. Recently, the effects of vitamin D have been recognized to play a central role not only in other common bone lesions such as osteoporosis due to aging and steroid induced osteoporosis, but in immune function and surveillance, growth and development, and cardiac and skeletal muscle function (19-22).
Several active forms of vitamin D have been identified, vitamin D receptors cloned, and nuclear binding proteins for the hormone identified and cloned (17-22). Studies on the regulation of 1 xcex1-hydroxylase activity are limited by the lack of a renal cell line with regulated expression of the enzyme. The only reports of 1-xcex1-hydroxylase activity in culture utilize freshly isolated chicken renal cortical cells in which the activity declines precipitously within 48 hours of plating in culture (28).
The importance of the renal 1-xcex1-hydroxylase is best understood by comparing the kinetics of the renal enzyme to other forms in the body (29-30). Demonstration that nephrectomy in pregnant rats did not completely abolish 1, 25-diOH-D3 formation sparked an intensive search for extrarenal sites of 1 xcex1-hydroxylase activity (29). Although 1 xcex1-hydroxylase activity has been reported in monocytes, liver, aortic endothelium and a variety of placental and fetal tissues, the enzyme kinetics contrast sharply with the renal 1 xcex1-hydroxylase. Extrarenal 1-xcex1-hydroxylase has a much higher Km indicating that much higher substrate levels are needed for activity (29). In the uremic patient, extrarenal 1,25-diOH D3 production is very limited due to a relative lack of substrate. Administrating large quantities of 25-OH D3 substrate to anephric patients modestly boosts plasma 1,25-diOH D3 levels (29).
The lack of a differentiated polarized line of renal tubular epithelial cells for investigative purposes persists despite extensive searches by several laboratories (31-38). Renally derived cell lines transformed with viruses or tumor cells to produce immortality continue as some of the most popular cell biological tools to study polarized delivery (31, 33, 35). But these renally derived immortal cell lines such as MDCK or LLP-CK1 retain few if any of the differentiated features characteristic of renal epithelial cells. Similarly, primary cultures rapidly dedifferentiate and modalities as diverse as basement membrane matrices, growth supplements or Millipore inserts achieve only modest degrees of polarity (37-38).
The pathognomonic structural features of renal proximal tubular epithelial cells are the abundance of apically derived microvilli, the glycoprotein content of associated intermicrovillar clefts, and the highly distinctive arrangement of subapical endosomal elements (39-40). Renal epithelial cells of the proximal tubule are characterized by thousands of long apical microvilli. The apical endosomal machinery begins in intermicrovillar clefts. The endosomal pathway is characterized by clathrin coated vesicles, small spherical endosomal vesicles, with deeper larger endosomal vacuoles (33, 39). From the endosomal vacuoles proteins and lipids either recycle to apical surface in dense apical tubules or shuttle to lysosomes to be degraded.
A cluster of apical proteins with homologous sequence repeats are especially desirable to express in cultured cells as they are thought to be molecular mediators of renal injury (41-43). Two of these proteins megalin (gp330) and cubulin (gp280) (Moestrup, et al., J. Biol. Chem.xcex2273 (9):5325-5242 (1998) are molecular mediators of tubular vacuolation and ensuing secondary damage. Megalin (gp330) is a receptor found on the luminal surface of the proximal tubular cells of the kidney. Megalin binds several proteins and drugs including aminoglycoside antibiotics and other polybasic drugs. Megalin is expressed in the kidney, lung, testes, ear, and placenta. The only cells which express megalin in culture are immortalized placental cells. There is no known renal cell culture which expresses megalin. Gp280 is a receptor found on the luminal surface of the proximal tubular cells of the kidney. Gp280 binds several proteins and drugs including intrinsic factor-cobalamin (vitamin B12 bound to its carrier protein) and myeloma light chains. Cubulin (gp280) is expressed in the kidney, ear, and placenta. The only cells which express cubulin (gp280) in culture are immortalized placental cells. There is no known renal cell culture which expresses cubulin (gp280).
Erythropoietin (EPO) is a hormone produced in the kidney, and secreted into the blood. Erythropoietin controls the rate of production of red blood cells by the bone marrow. Erythropoietin may be produced by the interstitial cells between the tubules or the proximal tubular cells or both. Erythropoietin production is lost in all known renal cell culture systems. Erythropoietin is mainly used to treat anemia in dialysis patients but is also popular to treat the anemia of AIDS patients and many forms of cancer.
The prior art is deficient in the lack of effective means of producing functional proteins including hormones in response to shear stress. Further, the prior art is deficient in the identification of shear stress response elements in epithelial cell genes. The present invention fulfills this longstanding need and desire in the art.
In one embodiment of the present invention, there is provided a method of producing a functional protein, comprising the steps of: isolating mammalian cells; placing said cells into a rotating wall vessel containing a cell culture comprising culture media and culture matrix; producing three-dimensional cell aggregates under simulated microgravity conditions; and detecting expression of the functional protein in the cell culture.
In another embodiment of the present invention, there is provided a method of inducing expression of at least one gene in a cell, comprising the steps of: contacting said cell with an transcription factor decoy oligonucleotide sequence directed against a nucleotide sequence encoding a shear stress response element; and determining the expression of said gene in said cell.
In yet another embodiment of the present invention, there is provided a transcription factor decoy, comprising an oligonucleotide sequence directed. against a nucleotide sequence encoding a shear stress response element.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.